PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a high incidence of life- threatening hospital-acquired infections. P. aeruginosa pathogenicity relies on the process of bacterial cell-cell communication known as quorum sensing. Quorum-sensing bacteria produce, release, and, as a coordinated group, detect the buildup of extracellular signal molecules, called autoinducers. Quorum sensing allows bacteria to count the members of the vicinal community and undertake collective behaviors only when there are enough cells present for group behaviors to effectively promote survival and/or infection. P. aeruginosa has two main quorum-sensing systems, each composed of an autoinducer synthase and an autoinducer receptor (LasI/LasR and RhlI/RhlR). A new protein, the thioesterase, PqsE, has been identified by the Bassler laboratory to possess an activity that allows the RhlR arm of the quorum-sensing system to be activated in the absence of the canonical, RhlI-produced autoinducer. Importantly, deletion of pqsE makes P. aeruginosa completely avirulent in nematode and mouse models of infection, as does deletion of rhlR. These findings reveal PqsE as an exciting new target for anti-microbial drug design to combat P. aeruginosa infections. The goals of this research are to 1) develop potent small molecule inhibitors of PqsE and 2) use inhibitor-bound PqsE structures to design point mutations to dissect the roles PqsE plays in quorum sensing. I will use multiple chemical screening strategies to discover molecules that inhibit PqsE function, optimize those lead molecules through rounds of synthetic diversification, and characterize their mechanisms of action. Crystallography of my inhibitors bound to PqsE will be used to inform the design of genetic tools to investigate how PqsE differentially regulates gene expression under planktonic and biofilm growth conditions. My work will provide an understanding of how PqsE contributes to quorum sensing in P. aeruginosa, and with new molecules in hand that interfere with the process, the field can develop strategies to treat deadly hospital-acquired infections. In order to accomplish the goals of this project, I will utilize techniques in chemistry, biochemistry, and structural biology, and I will learn techniques in microbiology and bacterial genetics. With the development of these skills over the course of the next three years, I aim to gain independence as an interdisciplinary researcher and project leader. This will prepare me for, and form the basis of running my own research program in an academic lab, beyond my postdoctoral work at Princeton University.